Anti-Mesothelin Antibodies

ABSTRACT

This invention relates to the use of monoclonal and polyclonal antibodies that specifically bind to and become internalized by mesothelin-positive cells and also induce an immune effector activity such as antibody dependent cellular cytotoxicity. The antibodies are useful in specific delivery of pharmacologic agents to mesothelin expressing cells as well as eliciting an immune-effector activity particularly on tumor cells and precursors. The invention is also related to cells expressing the monoclonal antibodies, polyclonal antibodies, antibody derivatives, such as human, humanized, and chimeric monoclonal antibodies, antibody fragments, mammalian cells expressing the monoclonal antibodies, derivatives and fragments, and methods of treating cancer using the antibodies, derivatives and fragments.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.11/373,546 filed Mar. 9, 2006, now allowed, which claims benefit of U.S.Provisional Application 60/660,177, filed Mar. 10, 2005, both of whichare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to antibodies that specifically bind tomesothelin. In some embodiments of the invention, the antibodies areeither internalized by cells expressing or bearing mesothelin(“mesothelin-positive cells”) or induce an immune effector activity onmesothelin-positive cells. The antibodies of the invention are useful inspecific delivery of pharmacologic agents to mesothelin-positive cellsas well as in eliciting an immune effector activity onmesothelin-positive cells, for example, tumor cells and precursors. Theinvention is also related to polynucleotides encoding the antibodies ofthe invention, cells expressing the antibodies of the invention, methodsof producing the antibodies of the invention, compositions of theantibodies, methods of inhibiting the growth of dysplastic cells usingthe antibodies, and methods of treating cancer using the antibodies.

BACKGROUND OF THE INVENTION

Pancreatic cancer is the fifth leading cause of death in the U.S. with a5-year survival rate of less than 5%. Although radiotherapy andchemotherapy are the recommended treatments and have enjoyed somesuccess, no current treatment effects 2-year survival for patients withlocally advanced and metastatic disease (Lawrence, Semin. Oncol.,22:68-71, 1995).

A difficulty that is commonly encountered when treating patients thathave pancreatic as well as other cancers with cytotoxic small moleculedrugs is that the cytotoxin causes toxicity to normal tissues as well ascancerous tissues. One approach to obtain higher specificity for thecancer tissue is the use of antibodies that can target specific antigensexpressed in cancer cells that are not expressed or are expressed at alower level on normal cells. These target antigens can be exploitedusing antibodies to specifically kill antigen-bearing tumor cells by avariety of mechanisms including inhibiting the biological activity ofthe antigen, eliciting an immune effector activity by complementdependent cytotoxicity (CDC) and/or antibody dependent cellularcytotoxicity (ADCC), or by delivering immuno- or radio-conjugates that,when delivered to the antigen-bearing cell, specifically kill the targetcell. Finding antibodies that can specifically bind to and effectivelykill antigen-bearing tumor cells has proven difficult for many cancers.This has been due in part to the inability to obtain robust tumor lysisdue to either a lack of immune effector function or of efficientinternalization of antibodies carrying immunotoxins. Due to theexpression profile for mesothelin in pathologic tissue, there is anopportunity to obtain tumor-specific targeting for several cancer typesincluding but not limited to pancreatic, ovarian, and lung cancer andmesothelioma.

Mesothelin is a glycosylphosphatidylinositol (GPI)-linked glycoproteinsynthesized as a 69 kDa precursor and proteolytically processed into a30 kDa NH₂-terminal secreted form and a 40 kDa membrane-bound form(Yamaguchi, et al. (1994) J. Biol. Chem. 269:805-808). Mesothelin ishighly expressed on the surface of pancreatic cancers, ovarian cancers,mesotheliomas, lung cancers, and some other cancers. Its expression islimited on normal tissues, making it a potential target for cancertherapy. (Cao, et al., Mod. Pathol. 14:2005; Hassan, et al., Clin.Cancer Res. 2004 Jun. 15; 10(12 Pt 1):3937-42).

Administration of antibodies against mesothelin has been proposed as astrategy for treatment of mesothelioma as well as lung, ovarian, andpancreatic cancer. Full-length antibodies against mesothelin that canelicit a robust immune-effector activity and internalize for delivery oftoxic conjugates, however, have not been previously developed.

In 1992, Chang et al. described monoclonal antibodies that recognizedantigens on human ovarian carcinoma cells (Chang, et al., Am. J. Surg.Pathol. 1992 16:259-68). This antibody, called K1, was chemicallyconjugated to a truncated form of Pseudomonas exotoxin and found to bindmesothelin-positive cells and cancer cells. However, it was not usefulas an immunotoxin conjugate due to its poor internalization ability.U.S. Pat. No. 6,083,502 describes mesothelin and uses for targeting anddiagnosing mesothelin-positive cells using antibody K1.

Subsequent single chain antibodies were produced that bound withhigh-affinity and had potent antitumor activity on mesothelin-positivetumors as a conjugate. One such single chain antibody is SSI(scFv)-PE38which has a high binding affinity (K_(d) of 0.7 nM) to mesothelin. Thissingle chain antibody is a stabilized form of the Fv in which adisulfide bond connects the light and heavy chain domains of the Fv. SS1(scFv)-PE38 has been shown to have activity in killing tumor cells byinternalization of the single chain antibody-immunotoxin complex(Hassan, et al., Clin. Cancer Res., 8: 3520-6, 2002; Hassan, et al.,Proc. Am. Soc. Clin. Oncol., 21: 29a, 2002). Other groups have alsodeveloped antibodies that can bind to mesothelin and foundoverexpression of this antigen to be associated with various cancers(Scholler, et al., Proc. Natl. Acad. Sci. U.S.A. 1999 Sep. 28;96(20):11531-6; Ordonez, Am. J. Surg. Pathol. 27:1418-28, 2003).

U.S. Pat. No. 6,809,184 describes a single chain high affinity antibodythat binds to mesothelin at a different epitope than the K1 antibody.This antibody fragment was found to internalize in mesothelin-positivecells as a single chain fragment linked to an immunotoxin. The antibodywas named SS1.

Attempts to develop immunoconjugated antibodies that can specificallytarget mesothelin have been performed with little success due to poorinternalization and/or affinity (Hassan, et al., J Immunother. 2000July-August; 23(4):473-9). This lack of internalization could be due tolow affinity or poor internalization due to antibody composition and/orepitope binding. In addition, generation of the monoclonal antibody(m-Ab) K1 as an immunoconjugate was attempted because the unconjugatedform was not cytotoxic itself (Hassan, et al., Clin. Cancer Res.,10:3937-3942, 2004).

Provided herein are in-out antibodies that can internalize inmesothelin-positive cells and elicit a cytotoxic effect via immuneeffector activity. Also provided are antibody therapies for cancer, inparticular for mesothelin-positive cancers, for example, pancreatic,ovarian, mesothelioma, and lung cancers, using antibodies that elicit arobust immune effector activity yet retain the ability to internalizeand facilitate the delivery of toxins to mesothelin-positive cells.

SUMMARY OF THE INVENTION

The invention provides mesothelin-specific antibodies that alternativelyelicit a robust immune-effector function or internalize inmesothelin-positive cells, referred to here as in-out mesothelinantibodies. As used herein, “in-out antibodies” (“in-out Abs”) refer toantibodies that can alternatively elicit an immune effector activity andinternalize within an antigen-presenting cell by binding to targetantigen. Without wishing to be bound by any particular theory, it isbelieved that in-out Abs bind to the cell surface of an antigen-positivecell and internalize after a period of time unless engaged byimmune-effector cells and/or biochemicals that are recruited to theantigen-antibody-positive cell. Methods for generating antibodies thatare able to elicit an immune effector effect such antibody-dependentcellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC)and to internalize have been previously described (Wolff, et al., CancerRes. 1993 Jun. 1; 53:2560-5), however, it is not obvious that in-outantibodies can be developed against any antigen or epitope (Kusano etal., Anticancer Res. 1993 November-December; 13(6A):2207-12). Antibodiesthat can target cell surface antigens that routinely internalize do notalways internalize upon binding to the cell surface antigen (Cogliati etal., Anticancer Res. 11:417-21, 1991). Moreover, antibodies that cantarget cell surface antigens do not always elicit an immune effectorfunction upon binding to the cell surface antigen (Niwa, et al., CancerRes. 64:2127-33, 2004; Kikuchi, et al., Leuk. Res. 29:445-50, 2005;Scott, et al., Cancer Immun. February 22; 5:3, 2005). In-out antibodiesthat can target mesothelin have not been described previously.

Provided herein are antibodies that bind to the cell surface antigenmesothelin and can, in the alternative, elicit an immune effectoractivity (e.g., ADCC or CDC) and internalize within antigen(mesothelin)-positive cells. These antibodies are useful for cancertherapy.

The invention provides antibodies that specifically bind to mesothelinwherein the antibodies are distinguished from MAb K1 in that (a) theantibodies bind with greater affinity and/or avidity than MAb K1; (b)the antibodies elicit an immune effector effect such as but not limitedto ADCC or CDC; and (c) as an alternative to (b), the antibodiesinternalize in mesothelin-positive cells.

The invention provides antibodies that specifically bind to mesothelinwherein the antibodies are distinguished from SS1 in that (a) theantibody internalizes in mesothelin-positive cells; and (b) in thealternative, the antibodies elicit an immune effector activity, such asbut not limited to ADCC or CDC. In some embodiments, the antibodies ofthe invention bind with a different affinity and/or avidity than SS1.

The antibodies of the invention include chimeric antibodies, including,but not limited to human-mouse chimeric antibodies. The antibodies ofthe invention may also be humanized antibodies. The antibodies of theinvention may also be fully human antibodies. The invention alsoprovides: hybridoma cells that express the antibodies of the invention;polynucleotides that encode the antibodies of the invention; vectorscomprising the polynucleotides that encode the antibodies of theinvention; and expression cells comprising the vectors of the invention,referred to as transfectomas.

The invention also provides methods of producing antibodies of theinvention. In some embodiments, the methods involve a step of culturinga transfectoma or hybridoma cell that expresses an antibody of theinvention. The antibody-producing cells of the invention may bebacterial, yeast, insect, or animal cells, and preferably, are mammaliancells.

The invention further provides methods of inhibiting the growth ofmesothelin-positive cells, such as but not limited to, tumor ordysplastic cells associated with increased expression of mesothelinrelative to normal cells, comprising administering to a subject withsuch cells a composition comprising an in-out antibody of the invention.The methods may be used for various malignant and dysplastic conditions,such as, but not limited to mesothelioma and pancreatic, ovarian andlung cancer. In preferred embodiments, the subjects are animals. In morepreferred embodiments, the subjects are mammals. In a most preferredembodiment, the subjects are human. In some embodiments, the antibodiesare conjugated to one or more chemotherapeutic agents such as, but notlimited to radionuclides, toxins, and cytotoxic and cytostatic agents.In other embodiments the antibodies are used in combination with one ormore chemotherapeutic agents. In-out antibodies can be administered as asingle agent, as a conjugated or unconjugated antibody, or incombination with the conjugated or unconjugated forms or anothertherapeutic agent.

Other features and advantages of the invention will be apparent from thedetailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of FACS analysis of MSAb-1 binding tomesothelin-expressing cells (OVCAR-3-ovarian and PANC 1-pancreaticcells) while no binding is observed on A549 cells. These data wereconfirmed by western blot analysis (not shown).

FIG. 2 demonstrates that MSAb-1 elicits a robust antibody dependentcellular cytotoxicity (ADCC) activity. Tumor cell line OVCAR-3 (referredto as target) which expresses mesothelin was incubated with humanperipheral blood lymphocytes (PBLs) alone (no Ab lane); with MSAb-1 andPBLs; or with control Ig and PBLs (normal IgG). Cell cultures wereassayed for killing by monitoring for lactate dehydrogenase (LDH)release that occurs upon cell lysis. As shown here, MSAb-1 has ADCCactivity on mesothelin-expressing cells.

FIG. 3 demonstrates that MSAb-1 internalizes in mesothelin-expressingcells. FIG. 3 shows the ability of MSAb-1 linked to saporin (triangles)to kill cells in contrast to MSAb-1 unconjugated (X) while an isotypecontrol antibody ML1 did not kill cells in conjugated or unconjugatedtoxin form (diamond and square, respectively). As control, cells notexpressing mesothelin were used. Toxin conjugated MSAb-1 has no toxiceffect in conjugated or unconjugated form on the control cells. Thesedata support the findings that MSAb-1 internalizes inmesothelin-positive cells.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The reference works, patents, patent applications, and scientificliterature, including accession numbers to GenBank database sequencesthat are referred to herein establish the knowledge of those with skillin the art and are hereby incorporated by reference in their entirety tothe same extent as if each was specifically and individually indicatedto be incorporated by reference. Any conflict between any referencecited herein and the specific teachings of this specification shall beresolved in favor of the latter. Likewise, any conflict between anart-understood definition of a word or phrase and a definition of theword or phrase as specifically taught in this specification shall beresolved in favor of the latter.

Standard reference works setting forth the general principles ofrecombinant DNA technology known to those of skill in the art includeAusubel et al. CURRENT PROTOCOLS 1N MOLECULAR BIOLOGY, John Wiley &Sons, New York (1998); Sambrook et al. MOLECULAR CLONING: A LABORATORYMANUAL, 2D ED., Cold Spring Harbor Laboratory Press, Plainview, N.Y.(1989); Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODSIN BIOLOGY AND MEDICINE, CRC Press, Boca Raton (1995); McPherson, Ed.,DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press, Oxford (1991).

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting. As used in this specificationand the appended claims, the singular forms “a”, “an” and “the” includeplural referents unless the content clearly dictates otherwise. Thus,for example, reference to “a cell” includes a combination of two or morecells, and the like.

Each range recited herein includes all combinations and sub-combinationsof ranges, as well as specific numerals contained therein.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

The invention provides methods for decreasing or inhibiting the growthof mesothelin-positive cells (e.g., cancer cells) and the progression ofneoplastic disease using in-out monoclonal antibodies that specificallybind to mesothelin. The methods of the invention may be used to modulatethe growth of mesothelin-positive cells and the progression of cancer inmammals, including humans. The mesothelin-positive cells that may beinhibited include all cancer cells that have an increased expression ofmesothelin in relation to normal human tissues, for example but notlimited to pancreatic cancer, ovarian cancer, mesothelioma, and lungcancer cells.

Without wishing to be bound by any particular theory of operation, it isbelieved that the increased expression of mesothelin in cancer cellsresults in an increased cell surface expression of the membrane boundform on the surface of the cells. Therefore, cancer cells have anincreased expression of mesothelin relative to normal tissues. Thus, themembrane-bound mesothelin is an ideal target for in-out antibody therapyin cancer.

As used herein, the term “epitope” refers to the portion of an antigento which a monoclonal antibody specifically binds and includes, forexample, conformational epitopes.

As used herein, the term “conformational epitope” refers to adiscontinuous epitope formed by a spatial relationship between aminoacids of an antigen other than a continuous or unbroken series of aminoacids.

As used herein, the term “immune effector activity” refers to anantibody that can kill cells by antibody dependent cellular cytotoxicity(ADCC) or complement dependent cytotoxicity (CDC).

As used herein, the term “in-out antibody” refers to an antibody thatcan, in the alternative, elicit an immune-effector activity andinternalize within the cell by binding to target antigen.

As used herein, the phrase “in the alternative” when referring to theability of an antibody to internalize or elicit an immune effectoractivity means that the antibody has the ability to both internalize andelicit an immune effector activity but cannot do both simultaneously.

As used herein, the term “inhibition of growth of dysplastic cells invitro” means a decrease in the number of tumor cells in culture by about5%, preferably about 10%, more preferably about 20%, more preferablyabout 30%, more preferably about 40%, more preferably about 50%, morepreferably about 60%, more preferably about 70%, more preferably about80%, more preferably about 90%, and most preferably about 100%. In vitroinhibition of tumor cell growth may be measured by assays known in theart, for example, the GEO cell soft agar assay.

As used herein, the term “inhibition of growth of dysplastic cells invivo” means a decrease in the number of tumor cells, in an animal, byabout 5%, preferably about 10%, more preferably about 20%, morepreferably about 30%, more preferably about 40%, more preferably about50%, more preferably about 60%, more preferably about 70%, morepreferably about 80%, more preferably about 90%, and most preferablyabout 100%. In vivo modulation of tumor cell growth may be measured byassays known in the art, for example but not limited to using theResponse Evaluation Criteria in Solid Tumors (RECIST) parameters(available online through the National Cancer Institute Cancer TherapyEvaluation Program).

As used herein, “dysplastic cells” refers to cells that exhibit abnormalgrowth properties, such as but not limited to growth in soft agar, lackof contact inhibition, failure to undergo cell cycle arrest in theabsence of serum, and formation of tumors when injected intoimmune-compromised mice. Dysplastic cells include, but are not limitedto tumors, hyperplasia, and the like.

The term “preventing” refers to decreasing the probability that anorganism contracts or develops an abnormal condition.

The term “treating” refers to having a therapeutic effect and at leastpartially alleviating or abrogating an abnormal condition in theorganism. Treating includes inhibition of tumor growth, maintenance ofinhibited tumor growth, and induction of remission.

“Therapeutic effect” refers to the reduction, elimination, or preventionof a disease or abnormal condition, symptoms thereof, or side effectsthereof in the subject.

“Effective amount” refers to an amount necessary to produce a desiredeffect. A “therapeutically effective amount” means the amount that, whenadministered to a subject for treating a disease, condition or disorder,is sufficient to effect treatment for that disease. A therapeutic effectrelieves to some extent one or more of the symptoms of the abnormalcondition. In reference to the treatment of abnormal conditions, atherapeutic effect can refer to one or more of the following: (a) anincrease or decrease in the proliferation, growth, and/ordifferentiation of cells; (b) inhibition (i.e., slowing or stopping) ofgrowth of tumor cells in vivo (c) promotion of cell death; (d)inhibition of degeneration; (e) relieving to some extent one or more ofthe symptoms associated with the abnormal condition; and (f) enhancingthe function of a population of cells. The antibodies described hereineffectuate the therapeutic effect alone or in combination withconjugates or additional components of the compositions of theinvention.

As used herein, the term “inhibits the progression of cancer” refers toan activity of a treatment that slows the modulation of neoplasticdisease toward end-stage cancer in relation to the modulation towardend-stage disease of untreated cancer cells.

As used herein, the term “neoplastic disease” refers to a conditionmarked by abnormal proliferation of cells of a tissue.

As used herein the term “biomolecule” refers to any molecule that can beconjugated to, coadministered with, administered before or afteradministering the antibody, or otherwise used in association with theantibody of the invention. Biomolecules include, but are not limited to,enzymes, proteins, peptides, amino acids, nucleic acids, lipids,carbohydrates, and fragments, homologs, analogs, or derivatives, andcombinations thereof. Examples of biomolecules include but are notlimited to interleukin-2, interferon alpha, interferon beta, interferongamma, rituxan, zevalin, herceptin, erbitux, and avastin. Thebiomolecules can be native, recombinant, or synthesized, and may bemodified from their native form with, for example, glycosylations,acetylations, phosphorylations, myristylations, and the like. The termbiomolecule as it is used herein is not limited to naturally occurringmolecules, and includes synthetic molecules having no biological origin.

As used herein, the term “cytotoxic” or “cytostatic” agent refers to anagent that reduces the viability or proliferative potential of a cell.Cytotoxic or cytostatic agents can function in a variety of ways toreduce cell viability or proliferation, for example, but not by way oflimitation, by inducing DNA damage, inducing cell cycle arrest,inhibiting DNA synthesis, inhibiting transcription, inhibitingtranslation or protein synthesis, inhibiting cell division, or inducingapoptosis. As used herein, the term “chemotherapeutic agent” refers tocytotoxic, cytostatic, and antineoplastic agents that preferentiallykill, inhibit the growth of, or inhibit the metastasis of neoplasticcells or disrupt the cell cycle of rapidly proliferating cells. Specificexamples of chemotherapeutic agents include, but are not limited to,radionuclides, pokeweed antiviral protein, abrin, ricin and each oftheir A chains, altretamine, actinomycin D, plicamycin, puromycin,gramicidin D, doxorubicin, colchicine, cytochalasin B, cyclophosphamide,emetine, maytansine, amsacrine, cisplastin, etoposide, etoposideorthoquinone, teniposide, daunorubicin, gemcitabine, doxorubicin,mitoxantraone, bisanthrene, Bleomycin, methotrexate, vindesine,adriamycin, vincristine, vinblastine, BCNU, taxol, tarceva, avastin,mitomycin, modified Pseudomonas enterotoxin A, calicheamicin,5-fluorouracil, cyclophosphamide and certain cytokines such as TNF-alphaand TNF-beta.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

“Recombinant” when used with reference, e.g., to a cell, or nucleicacid, protein, or vector, indicates that the cell, nucleic acid, proteinor vector, has been modified by the introduction of a heterologousnucleic acid or protein or the alteration of a native nucleic acid orprotein, or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found within thenative (non-recombinant) form of the cell or express native genes thatare otherwise abnormally expressed, under expressed or not expressed atall.

The phrase “nucleic acid” or “polynucleotide sequence” refers to asingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases read from the 5′ to the 3′ end. Nucleic acids canalso include modified nucleotides that permit correct read through by apolymerase and do not alter expression of a polypeptide encoded by thatnucleic acid, including, for example, conservatively modified variants.

“Polypeptide,” “peptide” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymers. Polypeptides of the invention, includingantibodies of the invention, include conservatively modified variants.One of skill will recognize that substitutions, deletions or additionsto a nucleic acid, peptide, polypeptide, or protein sequence whichalter, add or delete a single amino acid or a small percentage of aminoacids in the encoded sequence is a “conservatively modified variant”where the alteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention. The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (33). The term “conservative substitution” also includesthe use of a substituted amino acid in place of an unsubstituted parentamino acid provided that such a polypeptide also displays the requisitebinding activity.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. “Amino acid analog” refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups (e.g., norleucine) or modified peptidebackbones but retain the same basic chemical structure as a naturallyoccurring amino acid. “Amino acid mimetic” refers to a chemical compoundhaving a structure that is different from the general chemical structureof an amino acid but that functions in a manner similar to a naturallyoccurring amino acid.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission (see Table 1 below).Nucleotides, likewise, can be referred to by their commonly acceptedsingle-letter codes.

TABLE 1 SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyr L-tyrosine G GlyL-glycine F Phe L-phenylalanine M Met L-methionine A Ala L-alanine S SerL-serine I Ile L-isoleucine L Leu L-leucine T Thr L-threonine V ValL-valine P Pro L-proline K Lys L-lysine H His L-histidine Q GlnL-glutamine E Glu L-glutamic acid W Trp L-tryptophan R Arg L-arginine DAsp L-aspartic acid N Asn L-asparagine C Cys L-cysteine

It should be noted that all amino acid sequences are represented hereinby formulae whose left to right orientation is in the conventionaldirection of amino-terminus to carboxy-terminus.

As used herein, the term “in vitro” or “ex vivo” refers to an artificialenvironment and to processes or reactions that occur within anartificial environment, for example, but not limited to, test tubes andcell cultures. The term “in vivo” refers to a natural environment (e.g.,an animal or a cell) and to processes or reactions that occur within anatural environment.

“Pharmaceutically acceptable,” “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

The term “pharmaceutically acceptable carrier” refers to reagents,excipients, cells, compounds, materials, compositions, and/or dosageforms which are, within the scope of sound medical judgment, suitablefor use in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other complicationcommensurate with a reasonable benefit/risk ratio. As described ingreater detail herein, pharmaceutically acceptable carriers suitable foruse in the present invention include gases, liquids, and semi-solid andsolid materials.

Except when noted, “subject” or “patient” are used interchangeably andrefer to mammals such as human patients and non-human primates, as wellas experimental animals such as rabbits, dogs, cats, rats, mice, andother animals. Accordingly, “subject” or “patient” as used herein meansany mammalian patient or subject to which the compositions of theinvention can be administered. In some embodiments of the presentinvention, the patient will be suffering from an infectious orinflammatory disease. In some embodiments of the present invention, thepatient will have been diagnosed with cancer. In an exemplary embodimentof the present invention, to identify candidate patients for treatmentaccording to the invention, accepted screening methods are employed todetermine the status of an existing disease or condition in a subject orrisk factors associated with a targeted or suspected disease orcondition. These screening methods include, for example, examinations todetermine whether a subject is suffering from an infectious disease, aninflammatory disease, or cancer. These and other routine methods allowthe clinician to select subjects in need of therapy.

“Therapeutic compound” as used herein refers to a compound useful in theprophylaxis or treatment of a disease or condition such as cancer.

“Concomitant administration,” “concurrent administration,” or“co-administration” as used herein includes administration of the activeagents (e.g., MAbs, chemotherapeutic agents, biomolecules), inconjunction or combination, together, or before or after each other. Themultiple agent(s) may be administered by the same or by differentroutes, simultaneously or sequentially, as long as they are given in amanner sufficient to allow all agents to achieve effectiveconcentrations at the site of action. A person of ordinary skill in theart would have no difficulty determining the appropriate timing,sequence, and dosages of administration for particular drugs andcompositions of the present invention.

“Immunoglobulin” or “antibody” is used broadly to refer to both antibodymolecules and a variety of antibody-derived molecules and includes anymember of a group of glycoproteins occurring in higher mammals that aremajor components of the immune system. The term “antibody” is used inthe broadest sense and specifically covers monoclonal antibodies,antibody compositions with polyepitopic specificity, bispecificantibodies, diabodies, and single-chain molecules, as well as antibodyfragments (e.g., Fab, F(ab′)2, and Fv), so long as they exhibit thedesired biological activity. An immunoglobulin molecule includes antigenbinding domains, which each include the light chains and theend-terminal portion of the heavy chain, and the Fc region, which isnecessary for a variety of functions, such as complement fixation. Thereare five classes of immunoglobulins wherein the primary structure of theheavy chain, in the Fc region, determines the immunoglobulin class.Specifically, the alpha, delta, epsilon, gamma, and mu chains correspondto IgA, IgD, IgE, IgG and IgM, respectively. As used herein“immunoglobulin” or “antibody” includes all subclasses of alpha, delta,epsilon, gamma, and mu and also refers to any natural (e.g., IgA andIgM) or synthetic multimers of the four-chain immunoglobulin structure.Antibodies non-covalently, specifically, and reversibly bind an antigen.The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that can be present inminor amounts. For example, monoclonal antibodies may be produced by asingle clone of antibody-producing cells. Unlike polyclonal antibodies,monoclonal antibodies are monospecific (e.g., specific for a singleepitope of a single antigen). The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention can be made by the hybridoma method first described byKohler et al., Nature, 256: 495, 1975, or can be made by recombinant DNAmethods. The “monoclonal antibodies” can also be isolated from phageantibody libraries using the techniques described in Marks et al., J.Mol. Biol., 222: 581-597, 1991, for example.

Antibody-derived molecules comprise portions of intact antibodies thatretain antigen-binding specificity, and comprise, for example, at leastone variable region (either a heavy chain or light chain variableregion). Antibody-derived molecules, for example, include molecules suchas Fab fragments, Fab′ fragments, F(ab′)2 fragments, Fd fragments, F(v)fragments, Fabc fragments, Fd fragments, Fabc fragments, Sc antibodies(single chain antibodies), diabodies, individual antibody light chains,individual antibody heavy chains, chimeric fusions between antibodychains and other molecules, heavy chain monomers or dimers, light chainmonomers or dimers, dimers consisting of one heavy and one light chain,and the like. All classes of immunoglobulins (e.g., IgA, IgD, IgE, IgGand IgM) and subclasses thereof are included.

Antibodies can be labeled/conjugated to toxic or non-toxic moieties.Toxic moieties include, for example, bacterial toxins, viral toxins,radioisotopes, and the like. Antibodies can be labeled for use inbiological assays (e.g., radioisotope labels, fluorescent labels) to aidin detection of the antibody. Antibodies can also be labeled/conjugatedfor diagnostic or therapeutic purposes, e.g., with radioactive isotopesthat deliver radiation directly to a desired site for applications suchas radioimmunotherapy (Garmestani et al., Nucl. Med. Biol., 28: 409,2001), imaging techniques and radioimmunoguided surgery or labels thatallow for in vivo imaging or detection of specific antibody/antigencomplexes. Antibodies may also be conjugated with toxins to provide animmunotoxin (see, Kreitman, R. J. Adv. Drug Del. Rev., 31: 53, 1998).

With respect to antibodies, the term, “immunologically specific” refersto antibodies that bind to one or more epitopes of a protein ofinterest, but which do not substantially recognize and bind othermolecules in a sample containing a mixed population of antigenicbiological molecules.

“Chimeric” or “chimerized” antibodies (immunoglobulins) refer toantibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (Morrison et al., Proc. Natl. Acad. Sci.U.S.A., 81: 6851-6855, 1984).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiescan comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications aremade to further refine and optimize antibody performance. In general,the humanized antibody will comprise substantially all of at least one,and typically two, variable domains, in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

“Fully human” refers to an immunoglobulin, such as an antibody, wherethe whole molecule is of human origin or consists of an amino acidsequence identical to a human form of the antibody.

“Hybridoma” refers to the product of a cell-fusion between a culturedneoplastic lymphocyte and a primed B- or T-lymphocyte which expressesthe specific immune potential of the parent cell.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

Various patents and other publications are cited herein and throughoutthe specification, each of which is incorporated by reference herein inits entirety.

Antibodies

The in-out antibodies of the invention specifically bind mesothelin andexhibit, in the alternative, the ability to induce an immune effectoractivity and the ability to internalize in mesothelin-positive cells. Insome embodiments, the antibodies bind to the same epitope as mAb K1 orSS1. In other embodiments, the antibodies bind to an epitope other thanthat bound by mAbK1 or SS1.

Antibodies suitable for use in the method of the invention, include, forexample, monoclonal or polyclonal antibodies, fully human antibodies,human antibody homologs, humanized antibody homologs, chimericantibodies, singles chain antibodies, chimeric antibody homologs, andmonomers or dimers of antibody heavy or light chains or mixturesthereof. The antibodies of the invention may include intactimmunoglobulins of any isotype including types IgA, IgG, IgE, IgD, IgM(as well as subtypes thereof). The light chains of the immunoglobulinmay be kappa or lambda.

The in-out antibodies of the invention include portions of intactantibodies that retain antigen-binding specificity, for example, Fabfragments, Fab′ fragments, F(ab′)2 fragments, F(v) fragments, heavychain monomers or dimers, light chain monomers or dimers, dimersconsisting of one heavy and one light chain, and the like. Thus, antigenbinding fragments, as well as full-length dimeric or trimericpolypeptides derived from the above-described antibodies are themselvesuseful for exhibiting in-out activity.

Chimeric antibodies may be produced by recombinant DNA technology inwhich all or part of the hinge and constant regions of an immunoglobulinlight chain, heavy chain, or both, have been substituted for thecorresponding regions from another animal's immunoglobulin light chainor heavy chain. In this way, the antigen-binding portion of the parentmonoclonal antibody is grafted onto the backbone of another species'antibody. One approach, described in EP 0239400 to Winter et al.describes the substitution of one species' complementarity determiningregions (CDRs) for those of another species, such as substituting theCDRs from human heavy and light chain immunoglobulin variable regiondomains with CDRs from mouse variable region domains. These alteredantibodies may subsequently be combined with human immunoglobulinconstant regions to form antibodies that are human except for thesubstituted murine CDRs which are specific for the antigen. Methods forgrafting CDR regions of antibodies may be found, for example inRiechmann et al. (1988) Nature 332:323-327 and Verhoeyen et al. (1988)Science 239:1534-1536.

Humanized antibodies are produced by recombinant DNA technology, inwhich at least one of the amino acids of a human immunoglobulin light orheavy chain that is not required for antigen binding has beensubstituted for the corresponding amino acid from a nonhuman mammalianimmunoglobulin light or heavy chain. For example, if the immunoglobulinis a mouse monoclonal antibody, at least one amino acid that is notrequired for antigen binding is substituted using the amino acid that ispresent on a corresponding human antibody in that position. Withoutwishing to be bound by any particular theory of operation, it isbelieved that the “humanization” of the monoclonal antibody inhibitshuman immunological reactivity against the foreign immunoglobulinmolecule.

As a non-limiting example, a method of performing complementaritydetermining region (CDR) grafting may be performed by sequencing themouse heavy and light chains of the antibody of interest that binds tothe target antigen (e.g., mesothelin) and genetically engineering theCDR DNA sequences and imposing these amino acid sequences tocorresponding human V regions by site-directed mutagenesis. Humanconstant region gene segments of the desired isotype are added, and the“humanized” heavy and light chain genes are co-expressed in mammaliancells to produce soluble humanized antibody. A typical expression cellis a Chinese Hamster Ovary (CHO) cell. Suitable methods for creating thechimeric antibodies may be found, for example, in Jones et al. (1986)Nature 321:522-525; Riechmann (1988) Nature 332:323-327; Queen et al.(1989) Proc. Nat. Acad. Sci. USA 86:10029; and Orlandi et al. (1989)Proc. Natl. Acad. Sci. USA 86:3833.

Queen et al. (1989) Proc. Nat. Acad. Sci. USA 86:10029-10033 and WO90/07861 describe the preparation of a humanized antibody. Human andmouse variable framework regions were chosen for optimal proteinsequence homology. The tertiary structure of the murine variable regionwas computer-modeled and superimposed on the homologous human frameworkto show optimal interaction of amino acid residues with the mouse CDRs.This led to the development of antibodies with improved binding affinityfor antigen (which is typically decreased upon making CDR-graftedchimeric antibodies). Alternative approaches to making humanizedantibodies are known in the art and are described, for example, inTempest (1991) Biotechnology 9:266-271.

“Single chain antibodies” refer to antibodies formed by recombinant DNAtechniques in which immunoglobulin heavy and light chain fragments arelinked to the Fv region via an engineered span of amino acids. Variousmethods of generating single chain antibodies are known, including thosedescribed in U.S. Pat. No. 4,694,778; Bird (1988) Science 242:423-442;Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward etal. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

The antibodies of the invention include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody from bindingto its epitope. Examples of suitable derivatives include, but are notlimited to fucosylated antibodies and fragments, glycosylated antibodiesand fragments, acetylated antibodies and fragments, pegylated antibodiesand fragments, phosphorylated antibodies and fragments, and amidatedantibodies and fragments. The antibodies and derivatives thereof of theinvention may themselves be derivatized by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherproteins, and the like. In some embodiments of the invention, at leastone heavy chain of the antibody is fucosylated. In some embodiments, thefucosylation is N-linked. In some preferred embodiments, at least oneheavy chain of the antibody comprises a fucosylated, N-linkedoligosaccharide.

The antibodies of the invention include variants having single ormultiple amino acid substitutions, deletions, additions, or replacementsthat retain the biological properties (e.g., internalization, bindingaffinity or avidity, or immune effector activity) of the antibodies ofthe invention. The skilled person can produce variants having single ormultiple amino acid substitutions, deletions, additions or replacements.These variants may include, inter alia: (a) variants in which one ormore amino acid residues are substituted with conservative ornonconservative amino acids, (b) variants in which one or more aminoacids are added to or deleted from the polypeptide, (c) variants inwhich one or more amino acids include a substituent group, and (d)variants in which the polypeptide is fused with another peptide orpolypeptide such as a fusion partner, a protein tag or other chemicalmoiety, that may confer useful properties to the polypeptide, such as,for example, an epitope for an antibody, a polyhistidine sequence, abiotin moiety and the like. Antibodies of the invention may includevariants in which amino acid residues from one species are substitutedfor the corresponding residue in another species, either at theconserved or nonconserved positions. In another embodiment, amino acidresidues at nonconserved positions are substituted with conservative ornonconservative residues. The techniques for obtaining these variants,including genetic (suppressions, deletions, mutations, etc.), chemical,and enzymatic techniques, are known to the person having ordinary skillin the art. Antibodies of the invention also include antibody fragments.A “fragment” refers to polypeptide sequences which are preferably atleast about 40, more preferably at least to about 50, more preferably atleast about 60, more preferably at least about 70, more preferably atleast about 80, more preferably at least about 90, and more preferablyat least about 100 amino acids in length, and which retain somebiological activity or immunological activity of the full-lengthsequence, for example, mesothelin binding affinity or avidity, theability to internalize, and immune effector activity.

The invention also encompasses fully human antibodies such as thosederived from peripheral blood mononuclear cells of patients havingmesothelin-positive cancer cells, for example, ovarian cancer, lungcancer, mesothelioma, or pancreatic cancer patients. Such cells may befused with myeloma cells, for example, to form hybridoma cells producingfully human antibodies against mesothelin.

In preferred embodiments of the invention, the antibody comprises aheavy chain comprising an amino acid sequence of SEQ ID NO:2, SEQ IDNO:6, or SEQ ID NO:10:

SEQ ID NO 2: MSAb-1 heavy chain amino acid sequenceMGWSCIILFLVATATGVHSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK*SEQ ID NO 6 MSAb-2 heavy chain amino acidMGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK*SEQ ID NO: 10 MSAb-3 heavy chain amino acid sequenceMGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVKQAPGQGLEWIGLITPYNGASSYNQKFRGKATMTRDTSTSTVYMELSSLRSEDTAVYFCARGGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK*.

In some preferred embodiments, the heavy chain of the antibody isencoded by a nucleotide sequence comprising SEQ ID NO:1, SEQ ID NO:5, orSEQ ID NO:9:

SEQ. ID. NO. 1: MSAb-1 heavy chain nucleotide sequenceATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACAGCCAGGTACAACTGCAGCAGTCTGGGCCTGAGCTGGAGAAGCCTGGCGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGACTTATTACTCCTTACAATGGTGCTTCTAGCTACAACCAGAAGTTCAGGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGACCTCCTCAGTCTGACATCTGAAGACTCTGCAGTCTATTTCTGTGCAAGGGGGGGTTACGACGGGAGGGGTTTTGACTACTGGGGATCCGGGACCCCGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGG GAAATGA SEQ ID NO 5:MSAb-2 heavy chain nucleotide sequenceATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACAGCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGACTTATTACTCCTTACAATGGTGCTTCTAGCTACAACCAGAAGTTCAGGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGTTACGACGGGAGGGGTTTTGACTACTGGGGATCCGGGACCCCGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCTTATATTCAAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGG GAAATGA SEQ ID NO: 9MSAb-3 heavy chain nucleotide sequenceATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACAGCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGGCCCCTGGACAAGGGCTTGAGTGGATTGGACTTATTACTCCTTACAATGGTGCTTCTAGCTACAACCAGAAGTTCAGGGGCAAGGCCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTTCTGTGCGAGAGGGGGTTACGACGGGAGGGGTTTTGACTACTGGGGATCCGGGACCCCGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCTTATATTCAAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGG GAAATGA.

In some preferred embodiments, the antibodies of the invention comprisea light chain comprising an amino acid sequence of SEQ ID NO:4, SEQ IDNO:8, or SEQ ID NO: 12:

SEQ ID NO 4: MSAb-1-light chain amino acid sequenceMGWSCIILFLVATATGVHSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* SEQ ID NO: 8 MSAb-2 light chain aminoacid sequence MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* SEQ ID NO: 12 MSAb-3 light chain aminoacid sequence MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

In some preferred embodiments, the light chain of the antibody isencoded by a nucleotide sequence comprising SEQ ID NO:3, SEQ ID NO:7, orSEQ ID NO: 11:

SEQ ID NO 3: MSAb-1 light chain nucleotide sequenceATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACTCGGACATCGAGCTCACTCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCAGGTCGCTTCAGTGGCAGTGGGTCTGGAAACTCTTACTCTCTCACAATCAGCAGCGTGGAGGCTGAAGATGATGCAACTTATTACTGCCAGCAGTGGAGTAAGCACCCTCTCACGTTCGGATCCGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAAT SEQ ID NO: 7 MSAb-2light chain nucleotide sequenceATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACAGCGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGACACATCCAAACTGGCTTCTGGCGTCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTGGAGTAAGCACCCTCTCACGTTCGGATCCGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA SEQ ID NO: 11 MSAb-3light chain nucleotide sequenceATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACAGCGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGACACATCCAAACTGGCTTCTGGCGTCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTGGAGTAAGCACCCTCTCACGTTCGGATCCGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA.

In some embodiments of the invention, the antibody comprises a heavychain comprising an amino acid sequence of SEQ ID NO:2, 6, or 10 and alight chain comprising an amino acid sequence of SEQ ID NO:4, 8, or 12.In more preferred embodiments, the antibody comprises a heavy chaincomprising an amino acid sequence of SEQ ID NO:2 and a light chaincomprising an amino acid sequence of SEQ ID NO:4; a heavy chaincomprising an amino acid sequence of SEQ ID NO:6 and a light chaincomprising an amino acid sequence of SEQ ID NO:8; or a heavy chaincomprising an amino acid sequence of SEQ ID NO: 10 and a light chaincomprising an amino acid sequence of SEQ ID NO: 12.

The antibodies and derivatives thereof of the invention have bindingaffinities that include a dissociation constant (K) of less than 1×10⁻².In some embodiments, the K_(d) is less than 1×10⁻³. In otherembodiments, the K_(d) is less than 1×10⁻⁴. In some embodiments, theK_(d) is less than 1×10⁻⁵. In still other embodiments, the K_(d) is lessthan 1×10⁻⁶. In other embodiments, the K_(d) is less than 1×10⁻⁷. Inother embodiments, the K_(d) is less than 1×10⁻⁸. In other embodiments,the K_(d) is less than 1×10⁻⁹. In other embodiments, the K_(d) is lessthan 1×10⁻¹⁰. In still other embodiments, the K_(d) is less than 1×10¹¹.In some embodiments, the K_(d) is less than 1×10⁻¹². In otherembodiments, the K_(d) is less than 1×10⁻¹³. In other embodiments, theK_(d) is less than 1×10⁻¹⁴. In still other embodiments, the K_(d) isless than 1×10⁻¹⁵.

The antibodies of the invention may be used alone or with (e.g.,coadministered or conjugated to) a biomolecule or chemotherapeutic agentsuch as a cytotoxic or cytostatic agent. In some embodiments, thechemotherapeutic agent is a radionuclide, including, but not limited toLead-212, Bismuth-212, Astatine-211, Iodine-131, Scandium-47,Rhenium-186, Rhenium-188, Yttrium-90, Iodine-123, Iodine-125,Bromine-77, Indium-111, and fissionable nuclides such as Boron-10 or anActinide. In other embodiments, the chemotherapeutic agent is a toxin orcytotoxic drug, including but not limited to ricin, modified Pseudomonasenterotoxin A, calicheamicin, adriamycin, 5-fluorouracil, and the like.Methods of conjugation of antibodies and antibody fragments to suchagents are known in the literature.

Without wishing to be bound by any particular theory of operation, it isbelieved that the in-out antibodies of the invention are particularlyuseful to bind mesothelin due to an increased avidity of the antibody asboth “arms” of the antibody (Fab fragments) bind to separate mesothelinmolecules. This leads to a decrease in the dissociation (Kd) of theantibody and an overall increase in the observed affinity (K_(D)). Inaddition, antibodies of this invention bind to epitopes that allow forthe internalization of the antibody-antigen complex. These areespecially good features for targeting tumors as the antibodies of theinvention will bind preferentially to tumor tissue relative to normaltissue to attract immune cells and biomolecules for cytotoxicity and arecapable of internalizing for delivery of conjugated agents fortherapeutic effect.

Nucleic Acids

The invention also includes nucleic acids encoding the heavy chainand/or light chain of the anti-mesothelin antibodies of the invention.“Nucleic acid” or a “nucleic acid molecule” as used herein refers to anyDNA or RNA molecule, either single- or double-stranded and, ifsingle-stranded, the molecule of its complementary sequence in eitherlinear or circular form. In discussing nucleic acid molecules, asequence or structure of a particular nucleic acid molecule may bedescribed herein according to the normal convention of providing thesequence in the 5′ to 3′ direction. In some embodiments of theinvention, nucleic acids are “isolated.” This term, when applied to anucleic acid molecule, refers to a nucleic acid molecule that isseparated from sequences with which it is immediately contiguous in thenaturally occurring genome of the organism in which it originated. Forexample, an “isolated nucleic acid” may comprise a DNA molecule insertedinto a vector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a prokaryotic or eukaryotic cell or host organism. Whenapplied to RNA, the term “isolated nucleic acid” refers primarily to anRNA molecule encoded by an isolated DNA molecule as defined above.Alternatively, the term may refer to an RNA molecule that has beensufficiently separated from other nucleic acids with which it would beassociated in its natural state (i.e., in cells or tissues). An isolatednucleic acid (either DNA or RNA) may further represent a moleculeproduced directly by biological or synthetic means and separated fromother components present during its production.

Nucleic acids of the invention include nucleic acids having at least80%, more preferably at least about 90%, more preferably at least about95%, and most preferably at least about 98% homology to nucleic acids ofthe invention. The terms “percent similarity”, “percent identity” and“percent homology” when referring to a particular sequence are used asset forth in the University of Wisconsin GCG software program. Nucleicacids of the invention also include complementary nucleic acids. In someinstances, the sequences will be fully complementary (no mismatches)when aligned. In other instances, there may be up to about a 20%mismatch in the sequences.

Nucleic acids of the invention also include fragments of the nucleicacids of the invention. A “fragment” refers to a nucleic acid sequencethat is preferably at least about 10 nucleic acids in length, morepreferably about 40 nucleic acids, and most preferably about 100 nucleicacids in length. A “fragment” can also mean a stretch of at least about100 consecutive nucleotides that contains one or more deletions,insertions, or substitutions. A “fragment” can also mean the wholecoding sequence of a gene and may include 5′ and 3′ untranslatedregions.

The encoded antibody light chain preferably comprises an amino acidsequence of SEQ ID NO:4, 8, or 12. The encoded antibody heavy chainpreferably comprises an amino acid sequence of SEQ ID NO:2, 6, or 10. Insome embodiments of the invention, the heavy chain of the antibody isencoded by a nucleic acid comprising the nucleotide sequence of SEQ IDNO:1, 5, or 9. In some embodiments of the invention, the light chain ofthe anti-mesothelin antibody is encoded by a nucleic acid sequence ofSEQ ID NO:3, 7, or 11. In some embodiments of the invention are providednucleic acids encoding both a heavy chain and a light chain of anantibody of the invention. For example, a nucleic acid of the inventionmay comprise a nucleic acid sequence encoding an amino acid sequence ofSEQ ID NO:2, 6, or 10 and a nucleic acid sequence encoding an amino acidsequence of SEQ ID NO:4, 8, or 12.

Nucleic acids of the invention can be cloned into a vector. A “vector”is a replicon, such as a plasmid, cosmid, bacmid, phage, artificialchromosome (BAC, YAC) or virus, into which another genetic sequence orelement (either DNA or RNA) may be inserted so as to bring about thereplication of the attached sequence or element. A “replicon” is anygenetic element, for example, a plasmid, cosmid, bacmid, phage,artificial chromosome (BAC, YAC) or virus, that is capable ofreplication largely under its own control. A replicon may be either RNAor DNA and may be single or double stranded. In some embodiments, theexpression vector contains a constitutively active promoter segment(such as but not limited to CMV, SV40, Elongation Factor or LTRsequences) or an inducible promoter sequence such as the steroidinducible pIND vector (Invitrogen), where the expression of the nucleicacid can be regulated. Expression vectors of the invention may furthercomprise regulatory sequences, for example, an internal ribosomal entrysite. The expression vector can be introduced into a cell bytransfection, for example.

Nucleic acids encoding antibodies of the invention may be recombinantlyexpressed. The expression cells of the invention include any insectexpression cell line known, such as for example, Spodoptera frugiperdacells. The expression cell lines may also be yeast cell lines, such as,for example, Saccharomyces cerevisiae and Schizosaccharomyces pombecells. The expression cells may also be mammalian cells such as, forexample Chinese Hamster Ovary, baby hamster kidney cells, humanembryonic kidney line 293, normal dog kidney cell lines, normal catkidney cell lines, monkey kidney cells, African green monkey kidneycells, COS cells, and non-tumorigenic mouse myoblast G8 cells,fibroblast cell lines, myeloma cell lines, mouse NIH/3T3 cells, LMTKcells, mouse sertoli cells, human cervical carcinoma cells, buffalo ratliver cells, human lung cells, human liver cells, mouse mammary tumorcells, TR1 cells, MRC 5 cells, and FS4 cells. Nucleic acids of theinvention may be introduced into a cell by transfection, for example.Recombinantly expressed antibodies may be recovered from the growthmedium of the cells, for example.

Methods of Producing In-Out Antibodies to Mesothelin

Immunizing Animals

The invention also provides methods of producing in-out monoclonalantibodies that specifically bind to mesothelin. Antibodies of theinvention may be produced in vivo or in vitro. One strategy forgenerating antibodies against mesothelin involves immunizing animalswith mesothelin or cells expressing mesothelin. Animals so immunizedwill produce antibodies against the protein. Standard methods are knownfor creating monoclonal antibodies including, but are not limited to,the hybridoma technique (see Kohler & Milstein, (1975) Nature256:495-497); the trioma technique; the human B-cell hybridoma technique(see Kozbor et al. (1983) Immunol. Today 4:72) and the EBV hybridomatechnique to produce human monoclonal antibodies (see Cole, et al. inMONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., 1985, pp.77-96).

For in vivo antibody production, the antigen or antigen-positive cell isgenerally combined with an adjuvant to promote immunogenicity. Adjuvantsvary according to the species used for immunization. Examples ofadjuvants include, but are not limited to: Freund's complete adjuvant(“FCA”), Freund's incomplete adjuvant (“FIA”), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions), peptides, oil emulsions, keyhole limpethemocyanin (“KLH”), dinitrophenol (“DNP”), and potentially useful humanadjuvants such as Bacille Calmette-Guerin (“BCG”) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Immunization may be accomplished using well-known procedures. The doseand immunization regimen will depend on the species of mammal immunized,its immune status, body weight, and/or calculated surface area, etc.Typically, blood serum is sampled from the immunized mammals and assayedfor anti-mesothelin antibodies using appropriate screening assays asdescribed below, for example.

Mesothelin may be purified from cells or from recombinant systems usinga variety of well-known techniques for isolating and purifying proteins.For example, but not by way of limitation, mesothelin may be isolatedbased on the apparent molecular weight of the protein by running theprotein on an SDS-PAGE gel and blotting the proteins onto a membrane.Thereafter, the appropriate size band corresponding to mesothelin may becut from the membrane and used as an immunogen in animals directly, orby first extracting or eluting the protein from the membrane. As analternative example, the protein may be isolated by size-exclusionchromatography alone or in combination with other means of isolation andpurification. Other means of purification are available in such standardreference texts as Zola, MONOCLONAL ANTIBODIES: PREPARATION AND USE OFMONOCLONAL ANTIBODIES AND ENGINEERED ANTIBODY DERIVATIVES (BASICS: FROMBACKGROUND TO BENCH) Springer-Verlag Ltd., New York, 2000; BASIC METHODSIN ANTIBODY PRODUCTION AND CHARACTERIZATION, Chapter 11, “AntibodyPurification Methods,” Howard and Bethell, Eds., CRC Press, 2000;ANTIBODY ENGINEERING (SPRINGER LAB MANUAL.), Kontermann and Dubel, Eds.,Springer-Verlag, 2001.

Another strategy for generating in-out antibodies against mesothelininvolves immunizing animals with peptides corresponding to regions ofthe membrane bound form of mesothelin that allow for internalization ofantibodies that retain robust immune effector activity. Animals soimmunized will produce antibodies against the protein. Standard methodsare known for creating monoclonal antibodies including, but are notlimited to, the hybridoma technique (see Kohler & Milstein, (1975)Nature 256:495-497); the trioma technique; the human B-cell hybridomatechnique (see Kozbor et al. (1983) Immunol. Today 4:72) and the EBVhybridoma technique to produce human monoclonal antibodies (see Cole, etal. in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc.,1985, pp. 77-96).

Splenocytes from immunized animals may be immortalized by fusing thesplenocytes (containing the antibody-producing B cells) with an immortalcell line such as a myeloma line. Typically, myeloma cell line is fromthe same species as the splenocyte donor. In one embodiment, theimmortal cell line is sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). In someembodiments, the myeloma cells are negative for Epstein-Barr virus (EBV)infection. In preferred embodiments, the myeloma cells areHAT-sensitive, EBV negative and Ig expression negative. Any suitablemyeloma may be used. Murine hybridomas may be generated using mousemyeloma cell lines (e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines). These murine myeloma lines are available fromthe ATCC. These myeloma cells are fused to the donor splenocytespolyethylene glycol (“PEG”), preferably 1500 molecular weightpolyethylene glycol (“PEG 1500”). Hybridoma cells resulting from thefusion are selected in HAT medium which kills unfused and unproductivelyfused myeloma cells. Unfused splenocytes die over a short period of timein culture. In some embodiments, the myeloma cells do not expressimmunoglobulin genes.

Hybridomas producing a desired antibody which are detected by screeningassays such as those described below, may be used to produce antibodiesin culture or in animals. For example, the hybridoma cells may becultured in a nutrient medium under conditions and for a time sufficientto allow the hybridoma cells to secrete the monoclonal antibodies intothe culture medium. These techniques and culture media are well known bythose skilled in the art. Alternatively, the hybridoma cells may beinjected into the peritoneum of an unimmunized animal. The cellsproliferate in the peritoneal cavity and secrete the antibody, whichaccumulates as ascites fluid. The ascites fluid may be withdrawn fromthe peritoneal cavity with a syringe as a rich source of the monoclonalantibody.

Another non-limiting method for producing human antibodies is describedin U.S. Pat. No. 5,789,650 which describes transgenic mammals thatproduce antibodies of another species (e.g., humans) with their ownendogenous immunoglobulin genes being inactivated. The genes for theheterologous antibodies are encoded by human immunoglobulin genes. Thetransgenes containing the unrearranged immunoglobulin encoding regionsare introduced into a non-human animal. The resulting transgenic animalsare capable of functionally rearranging the transgenic immunoglobulinsequences and producing a repertoire of antibodies of various isotypesencoded by human immunoglobulin genes. The B-cells from the transgenicanimals are subsequently immortalized by any of a variety of methods,including fusion with an immortalizing cell line (e.g., a myeloma cell).

In-out antibodies against mesothelin may also be prepared in vitro usinga variety of techniques known in the art. For example, but not by way oflimitation, fully human monoclonal antibodies against mesothelin may beprepared by using in vitro-primed human splenocytes (Boerner et al.(1991) J. Immunol. 147:86-95).

Alternatively, for example, the antibodies of the invention may beprepared by “repertoire cloning” (Persson et al. (1991) Proc. Nat. Acad.Sci. USA 88:2432-2436; and Huang and Stollar (1991) J. Immunol. Methods141:227-236). Further, U.S. Pat. No. 5,798,230 describes preparation ofhuman monoclonal antibodies from human B antibody-producing B cells thatare immortalized by infection with an Epstein-Barr virus that expressesEpstein-Barr virus nuclear antigen 2 (EBNA2). EBNA2, required forimmortalization, is then inactivated resulting in increased antibodytiters.

In another embodiment, in-out antibodies against mesothelin are formedby in vitro immunization of peripheral blood mononuclear cells(“PBMCs”). This may be accomplished by any means known in the art, suchas, for example, using methods described in the literature (Zafiropouloset al. (1997) J. Immunological Methods 200:181-190).

In some embodiments of the invention, the procedure for in vitroimmunization is supplemented with directed evolution of the hybridomacells in which a dominant negative allele of a mismatch repair gene suchas PMS1, PMS2, PMS2-134, PMSR2, PMSR3, MLH1, MLH2, MLH3, MLH4, MLH5,MLH6, PMSL9, MSH1, and MSH2 is introduced into the hybridoma cells afterfusion of the splenocytes, or to the myeloma cells before fusion. Cellscontaining the dominant negative mutant will become hypermutable andaccumulate mutations at a higher rate than untransfected control cells.A pool of the mutating cells may be screened for clones that producehigher affinity antibodies, or that produce higher titers of antibodies,or that simply grow faster or better under certain conditions. Thetechnique for generating hypermutable cells using dominant negativealleles of mismatch repair genes is described in U.S. Pat. No.6,146,894, issued Nov. 14, 2000. Alternatively, mismatch repair may beinhibited using the chemical inhibitors of mismatch repair described byNicolaides et al. in WO 02/054856 “Chemical Inhibitors of MismatchRepair” published Jul. 18, 2002. The technique for enhancing antibodiesusing the dominant negative alleles of mismatch repair genes or chemicalinhibitors of mismatch repair may be applied to mammalian expressioncells expressing cloned immunoglobulin genes as well. Cells expressingthe dominant negative alleles can be “cured” in that the dominantnegative allele can be turned off, if inducible, eliminated from thecell and the like such that the cells become genetically stable oncemore and no longer accumulate mutations at the abnormally high rate.

Screening for In-Out Antibody Specificity

Screening for in-out antibodies that specifically bind to mesothelin maybe accomplished using an enzyme-linked immunosorbent assay (ELISA) inwhich microtiter plates are coated with immunizing antigen (wholeprotein or peptides). Antibodies from positively reacting clones can befurther screened for reactivity in an ELISA-based assay to mesothelinusing microtiter plates coated with mesothelin. Clones that produceantibodies that are reactive to mesothelin are selected for furtherexpansion and development. These antibodies can be further shown formesothelin specific binding using FACS analysis.

Confirmation of mesothelin reactive in-out antibodies exhibiting may beaccomplished, for example, using a standard immune effector assay tomonitor antibody dependent cellular cytotoxicity (ADCC). Mesothelinspecific antibodies exhibiting ADCC activity can then be conjugated witha fluorochrome or prodrug to monitor ability to internalize byvisualization or toxicity that occurs when prodrug is internalized andliberated from the antibody leading to the presence of the toxin.

Pharmaceutical Compositions of Antibodies

Another aspect of the invention features a pharmaceutical composition ofanti-mesothelin antibodies of the invention. The pharmaceuticalcompositions may be used to inhibit or reduce growth ofmesothelin-positive cells in vitro or in vivo. For example, thecompositions may be administerd to a patient to inhibit or reduce growthof tumor cells. In certain embodiments, the pharmaceutical compositionis formulated for administration by injection or infusion.

Pharmaceutical compositions of the invention may further comprise one ormore chemotherapeutic agents and/or biomolecules. In some embodiments,the antibody is conjugated to the chemotherapeutic agent or biomolecule.Suitable chemotherapeutic agents include but are not limited to aradionuclide, including, but not limited to Lead-212, Bismuth-212,Astatine-211, Iodine-131, Scandium-47, Rhenium-186, Rhenium-188,Yttrium-90, Iodine-123, Iodine-125, Bromine-77, Indium-111, andfissionable nuclides such as Boron-10 or an Actinide. In otherembodiments, the agent is a toxin or cytotoxic drug, including but notlimited to ricin, modified Pseudomonas enterotoxin A, calicheamicin,adriamycin, 5-fluorouracil, and the like.

Pharmaceutical compositions of the invention may be formulated with apharmaceutically acceptable carrier or medium. Suitable pharmaceuticallyacceptable carriers include water, PBS, salt solution (such as Ringer'ssolution), alcohols, oils, gelatins, and carbohydrates, such as lactose,amylose, or starch, fatty acid esters, hydroxymethylcellulose, andpolyvinyl pyrrolidine. Such preparations can be sterilized, and ifdesired, mixed with auxiliary agents such as lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, and coloring. Pharmaceutical carriers suitable foruse in the present invention are known in the art and are described, forexample, in Pharmaceutical Sciences (17^(th) Ed., Mack Pub. Co., Easton,Pa.).

Kits

According to yet another aspect of the invention, a kit is provided forinhibiting or reducing growth of mesothelin-positive cells, e.g., tumorcells, in vitro or in vivo. Also provided are kits for identifying thepresence of mesothelin-positive cells in vitro or in vivo.

The kits of the invention comprise an antibody or an antibodycomposition of the invention and instructions for using the kit in amethod for inhibiting or reducing growth of mesothelin-positive cells orin a method for identifying the presence of mesothelin-positive cells,for example, in a biological sample. The kit may comprise at least onechemotherapeutic reagent. The kit may comprise at least one biomolecule.The kit may comprise at least one diagnostic reagent. An example of adiagnostic reagent is a detectable label, for example but not limited toa radioactive, fluorescent, or chromophoric agent (e.g., ¹¹¹In-DOTA).The detectable label may comprise an enzyme. The kit may compriseinstructions and/or means for administering the antibody or antibodycomposition, for example, by injection or infusion.

Methods of Detecting a Mesothelin-Positive Cell

The methods of the invention include methods of detectingmesothelin-positive cells, including but not limited to dysplastic orcancer cells presenting mesothelin on the surface, such as but notlimited to ovarian, pancreatic, lung, or mesothelioma cancer cells. Themethod may be performed in vitro on a biological sample or in vivo.Methods of detecting mesothelin-positive cells according to theinvention comprise contacting anti-mesothelin antibody of the inventionwith a biological sample or administering anti-mesothelin antibody ofthe invention to a patient, wherein the antibody is labeled with adetectable label, for example but not limited to a radioactive,fluorescent, or chromophoric agent (e.g., ¹¹¹In-DOTA), and determiningbinding of the antibody to cells. Dysplastic or cancer cells associatedwith increased mesothelin expression will preferably exhibit increasedantibody binding relative to normal cells. The detectable label may bean enzyme.

Methods of Reducing the Growth of Mesothelin-Positive Cells

The methods of the invention are suitable for use in humans andnon-human animals identified as having mesothelin-positive cells, forexample, subjects identified as having a neoplastic condition associatedwith an increased expression of mesothelin. Non-human animals whichbenefit from the invention include pets, exotic (e.g., zoo animals) anddomestic livestock. Preferably the non-human animals are mammals.

The invention is suitable for use in a human or animal patient that isidentified as having a dysplastic disorder that is marked by increasedexpression of mesothelin in the neoplasm in relation to normal tissues.Once such a patient is identified as in need of treatment for such acondition, the method of the invention may be applied to effecttreatment of the condition. Dysplastic tissues that may be treatedinclude, but are not limited to ovary, lung, pancreas, and prostate.

The in-out antibodies and derivatives thereof for use in the inventionmay be administered orally in any acceptable dosage form such ascapsules, tablets, aqueous suspensions, solutions or the like. Theantibodies and derivatives thereof may also be administeredparenterally, for example, via the following routes of administration:subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intranasal, topically, intrathecal,intrahepatic, intralesional, and intracranial injection or infusiontechniques. Generally, the antibodies and derivatives will be providedas an intramuscular or intravenous injection.

The in-out antibodies and derivatives of the invention may beadministered alone or with a pharmaceutically acceptable carrier,including acceptable adjuvants, vehicles and excipients, for example,phosphate buffered saline

The effective dosage will depend on a variety of factors and it is wellwithin the purview of a skilled physician to adjust the dosage for agiven patient according to various parameters such as body weight, thegoal of treatment, the highest tolerated dose, the specific formulationused, the route of administration and the like. Generally, dosage levelsof between about 0.001 and about 100 mg/kg body weight per day of theantibody or derivative thereof are suitable. In some embodiments, thedose will be about 0.1 to about 50 mg/kg body weight per day of theantibody or derivative thereof. In other embodiments, the dose will beabout 0.1 mg/kg body weight/day to about 20 mg/kg body weight/day. Instill other embodiments, the dose will be about 0.1 mg/kg bodyweight/day to about 10 mg/kg body weight/day. Dosing may be as a bolusor an infusion. Dosages may be given once a day or multiple times in aday. Further, dosages may be given multiple times of a period of time.In some embodiments, the doses are given every 1-14 days. In someembodiments, the antibodies or derivatives thereof are given as a doseof about. 3 to 1 mg/kg i.p. In other embodiments, the antibodies ofderivatives thereof are provided at about 5 to 12.5 mg/kg i.v. In stillother embodiments, the antibodies or derivatives thereof are providedsuch that a plasma level of at least about 1 ug/ml is maintained.

Effective treatment may be assessed in a variety of ways. In oneembodiment, effective treatment is determined by a slowed progression oftumor growth. In other embodiments, effective treatment is marked byshrinkage of the tumor (i.e., decrease in the size of the tumor). Inother embodiments, effective treatment is marked by inhibition ofmetastasis of the tumor. In still other embodiments, effective therapyis measured by increased well-being of the patient including such signsas weight gain, regained strength, decreased pain, thriving, andsubjective indications from the patient of better health.

The antibodies of the invention may be administered before, after, orsimultaneously with another therapeutic or diagnostic agent. Forexample, the in-out antibodies of the invention may be administeredalone or with a cytotoxic agent such as but not limited to adriamycin,doxorubicin, gemcitabine, or 5-fluorouracil. The in-out antibodies ofthe invention may be administered alone or with a cytostatic agent suchas but not limited to tarceva and avastin. The in-out antibodies andderivatives of the invention may be administered alone or with a vaccineagent. The in-out antibodies and derivatives of the invention may beadministered alone or with another biomolecule such as but not limitedto interleukin-2, interferon alpha, interferon beta, interferon gamma,rituxan, zevalin, herceptin, erbitux, avastin.

The in-out antibodies and derivatives of the invention may beadministered as a homogeneous mixture of unconjugated or conjugatedantibody or as a heterogeneous mixture of unconjugated and conjugatedin-out antibody.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof.

EXAMPLES Example 1 In-Out Antibodies that can Bind to Mesothelin

The monoclonal antibody MSAb-1 was developed by cloning the variabledomain of a mesothelin FAb fragment to the human IgG1 constant region.The antibody was shown to bind specifically to mesothelin protein andcancer cells expressing mesothelin and was found to have a bindingconstant of 2 nM using BIACORE. MSAb-2 and MSAb-3 are variants of MSAb-1that have different nucleotide and amino acid sequences within theirrespective variable regions. To demonstrate mesothelin-specific binding,ELISA assays were performed using recombinant mesothelin in a 96-wellformat following methods used by those skilled in the art. Antibodiesfound to react by ELISA were further analyzed for mesothelin bindingusing FACS analysis following the manufacturer's protocol. Shown in FIG.1 are representative data of the FACS analysis wherebymesothelin-expressing ovarian and pancreatic tumor cells were positivefor MSAb-1 binding in contrast to null cells (A549).

Example 2 Immune Effector Activity of MSAb-1

Activity of MSAb-1 in-out antibody for immune effector activity wasassessed by standard antibody dependent cellular cytotoxicity (ADCC)assays on the mesothelin expressing OVCAR-3 cell line. Briefly, OVCAR-3target cells were seeded in flat-bottom 96-well microplates in completegrowth medium (RPMI-1640 containing 10% FBS, 2 mM L-glutamine). Thefollowing day, the complete medium was replaced with 100 μl of CHO-CDserum-free medium (Sigma) and 50 μl of antibody-containing conditionedmedium was added to target cells and incubated for 20 minutes at 37° C.Subsequently, 100 μl of serum-free medium containing 2×10⁵ of effectorcells were added to each well and cells were incubated for 5-6 hours at37° C., 5% CO₂. Effector cells were derived from human peripheral bloodmononuclear cells (PBMCs) isolated from healthy donors (purchased fromInterstate Blood Bank). Prior to use in ADCC assays, PBMCs wereactivated by seeding PBMCs at 2.5×10⁶/ml in complete RPMI containing 10ng/ml human recombinant interleukin 2 (R&D Systems) for 3 days at 37°C., 5% CO₂. Activated PBMCs were then added to OVCAR-3 cells at aneffector:target cell ratio of 5:1 and cultures were incubated for 5-6hours at 37° C., 5% CO₂. Supernatant was then collected from each welland transferred into ELISA plates and analyzed for ADCC activity asfollows. ADCC activity was monitored by lactate dehydrogenase (LDH)release, an endogenous enzyme used to measure ADCC in standard assays.LDH was monitored by adding 100 μl of LDH substrate (Roche), a chemicalthat when converted by LDH is spectrophotometrically detectable atOD₄₉₀, to supernatant and incubated for 10 minutes at ambienttemperature. LDH activity is proportional to the extent of the LDHenzyme released from lysed target cells. Optical density at 490 nm(OD₄₉₀) was obtained spectrophotometrically. 2% Triton X was added totarget cells alone as a “max” positive control, while target cells withPBMC and no antibody served as the “spontaneous” negative control. LDHvalues were obtained and percent of cytotoxicity was determined with theformula: (sample value−spontaneous)/(max−spontaneous)×100%, where‘spontaneous’=target cell lysis in absence of effector cells, and‘max’=target cell lysis in the presence of 2% Triton. Cytotoxicityelicited by 100 ng/ml of MORAb-A92 (protein A purified), an isotypecontrol antibody, was used as positive control. Non-specificcytotoxicity was monitored using 100 ng/ml of normal human IgG1antibody. The ratio obtained dividing the % cytotoxicity by theconcentration of the antibody for each well/clone (i.e.ratio=50(%)/100(ng/ml)=0.5) was set as the criterion for selecting leadclones with potentially enhanced effector function.

Analysis of MSAb-1 shows the ability to enhance ADCC activity (p=0.018)over cells incubated with control Ig or no antibody (FIG. 2). These datasupport the finding that MSAb-1 has cytotoxic effects via immuneeffector function.

Example 3 Internalization of MSAb-1

MSAb-1 internalizes when bound to mesothelin-expressing cells. Thisfinding is shown in FIG. 3 using the Hum-ZAP assay. Second immunotoxinsare conjugations of a secondary antibody to the ribosome inactivatingprotein saporin. If the primary antibody being tested is internalized,the saporin is transported into the cell via its binding to thesecondary antibody. Once internalized, saporin separates from its IgGconjugate, inhibits protein synthesis, and ultimately causes cell death.Hum-ZAP (Advanced Targeting Systems, cat# IT-22) is a secondary chemicalconjugate of affinity purified goat anti-human IgG, (mw 210 kDa) thatrecognizes human monoclonal antibodies. The control molecule, GoatIgG-SAP (Advanced Targeting Systems cat#IT-19) is a conjugate of normalgoat IgG and saporin. Briefly, cells were plated into flat-bottom96-well tissue culture plates at 2500/well in 80 μl of RPMI 1640 with10% FCS, 2.0 mM glutamine, 1.0 mM sodium pyruvate, and 0.1 mM MEMnon-essential amino acids. Twenty-four hours later, 10 μl of primaryantibodies ML-1 or MSAb-1 were added along with 101 of Hum-ZAP or GoatIgG-SAP to bring the total volume to 100 μl. Experiments were set upwith antibody titrations and include primary and secondary antibodiesalone as control. Four days later, cell viability was evaluated usingPromega CellTiter® Cytotoxicity Assay (cat# G3581) which reads viablecell number by spectrophotometry. All tests were performed intriplicate. Data was evaluated by comparing treated and untreated wellsand results are expressed as percent of control. As shown in FIG. 3,OVCAR-3 cells, which overexpress mesothelin (triangle) die upontreatment with toxin-conjugated MSAb-1 as compared to toxin-conjugatedand unconjugated isotype control ML1 antibody (diamond and square,respectively) and to unconjugated MSAb-1 (X). In contrast, MSAb-1 toxinconjugate has no cytotoxicity on mesothelin-null TP cells as compared topositive control ML 1 conjugated antibody which recognizes a cellsurface antigen expressed on TP cells.

SUMMARY

In summary, anti-mesothelin antibodies of the invention are capable ofeliciting ADCC yet internalize in mesothelin-positive cells. Theantibodies of the invention are useful for the treatment ofmesothelin-positive tumor cells either as a single agent or incombination therapy.

1. A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and an antibody that specifically binds to mesothelin and iscapable of both eliciting an immune effector activity and internalizingwithin mesothelin-positive cells, the antibody comprising a heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO: 2absent amino acids 1 to 19 thereof and a light chain variable domaincomprising the amino acid sequence of SEQ ID NO: 4 absent amino acids 1to 19 thereof; a heavy chain variable domain comprising the amino acidsequence of SEQ ID NO: 6 absent amino acids 1 to 19 thereof and a lightchain variable domain comprising the amino acid sequence of SEQ ID NO: 8absent amino acids 1 to 19 thereof; or a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 10 absent amino acids 1to 19 thereof and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO: 12 absent amino acids 1 to 19 thereof. 2.The pharmaceutical composition of claim 1 comprising a chemotherapeuticagent.
 3. The pharmaceutical composition of claim 1 comprising abiomolecule.
 4. A method of inhibiting the growth of mesothelin-positivedysplastic cells comprising administering to a subject having themesothelin-positive dysplastic cells an antibody that specifically bindsto mesothelin and is capable of both eliciting an immune effectoractivity and internalizing within mesothelin-positive cells, theantibody comprising a heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO: 2 absent amino acids 1 to 19 thereof and alight chain variable domain comprising the amino acid sequence of SEQ IDNO: 4 absent amino acids 1 to 19 thereof; a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 6 absent amino acids 1to 19 thereof and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO: 8 absent amino acids 1 to 19 thereof; or aheavy chain variable domain comprising the amino acid sequence of SEQ IDNO: 10 absent amino acids 1 to 19 thereof and a light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 12 absent aminoacids 1 to 19 thereof.
 5. The method of claim 4 wherein the dysplasticcells are ovarian carcinoma cells.
 6. The method of claim 4 wherein thedysplastic cells are pancreatic adenocarcinoma cells.
 7. The method ofclaim 4 wherein the dysplastic cells are mesothelioma cells.
 8. Themethod of claim 4 wherein the dysplastic cells are lung carcinoma cells.9. The method of claim 4 wherein the dysplastic cells are prostatecancer cells.
 10. The method of claim 4 wherein the subject is a human.11. The method of claim 4 wherein the antibody is conjugated to achemotherapeutic agent.
 12. The method of claim 4 further comprisingadministering to the subject a chemotherapeutic agent.
 13. The method ofclaim 4 wherein the antibody is administered as an unconjugatedantibody.
 14. The method of claim 4 wherein the antibody is administeredas a mixture of unconjugated antibody and antibody conjugated to achemotherapeutic agent.
 15. An isolated nucleic acid encoding a heavychain of an antibody that specifically binds to mesothelin, wherein theheavy chain comprises an amino acid sequence of SEQ ID NO: 2 absentamino acids 1 to 19 thereof, SEQ ID NO: 6 absent amino acids 1 to 19thereof, or SEQ ID NO: 10 absent amino acids 1 to 19 thereof.
 16. Theisolated nucleic acid of claim 15 comprising a nucleic acid sequence ofSEQ ID NO: 1 absent nucleotides 1 to 57 thereof, SEQ ID NO: 5 absentnucleotides 1 to 57 thereof, or SEQ ID NO: 9 absent nucleotides 1 to 57thereof, respectively.
 17. A vector comprising the isolated nucleic acidof claim
 15. 18. A cell comprising the isolated nucleic acid of claim15.
 19. A isolated nucleic acid encoding a light chain of an antibodythat specifically binds to mesothelin, wherein the light chain comprisesan amino acid sequence of SEQ ID NO: 4 absent amino acids 1 to 19thereof, SEQ ID NO: 8 absent amino acids 1 to 19 thereof, or SEQ ID NO:12 absent amino acids 1 to 19 thereof.
 20. The isolated nucleic acid ofclaim 19 comprising a nucleic acid sequence of SEQ ID NO: 3 absentnucleotides 1 to 57 thereof, SEQ ID NO: 7 absent nucleotides 1 to 57thereof, or SEQ ID NO: 11 absent nucleotides 1 to 57 thereof,respectively.
 21. A vector comprising the isolated nucleic acid of claim19.
 22. A cell comprising the isolated nucleic acid of claim
 19. 23. Acell that produces an antibody comprising a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 2 absent amino acids 1to 19 thereof and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO: 4 absent amino acids 1 to 19 thereof; aheavy chain variable domain comprising the amino acid sequence of SEQ IDNO: 6 absent amino acids 1 to 19 thereof and a light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 8 absent aminoacids 1 to 19 thereof; or a heavy chain variable domain comprising theamino acid sequence of SEQ ID NO: 10 absent amino acids 1 to 19 thereofand a light chain variable domain comprising the amino acid sequence ofSEQ ID NO: 12 absent amino acids 1 to 19 thereof.
 24. The cell of claim23, wherein the cell is a hybridoma.